2K1C operation does not alter cGMP synthesis or cGMP sensitivity in WT kidneys.

<p><b>A</b>, NO-stimulated cGMP-forming activity (DEA-NO 100 µM) determined in kidney homogenates of sham- and 2K1C-operated WT mice (n=6 mice per group). <b>B</b>, quantification of the β₁ subunit content with respect to the subunit amount in sham-operated WT mice using subunit specific antibodies (n=12 mice per group). <b>C</b>, concentration-response curves for 8-pCPT-cGMP of NA-contracted isolated perfused kidneys of sham- and 2K1C-operated WT mice in the presence of L-NAME (n=13 and 7 mice, respectively). Experiments were performed using the non-clipped kidney of 2K1C mice and the respective kidney of sham mice.</p

Evidence of enhanced eNOS-catalyzed NO formation induced by the 2K1C operation.

<p><b>A</b>, Western blot detection of p-eNOS (serine 1177) and eNOS in 50 µg kidney homogenates of sham- and 2K1C-operated WT mice and quantification with respect to the ratio of p-eNOS to eNOS in kidneys of sham-operated WT (n=8 and 7 mice, respectively). * P< 0.05 versus WT-sham, unpaired Student's t test. <b>B</b>, concentration-response curves for angiotensin II-induced vasoconstriction of isolated perfused kidneys of sham- and 2K1C-operated WT mice in the presence of L-NAME (n=9 and 6 mice, respectively). * P< 0.05, and ** P< 0.01 versus WT-sham, unpaired Student's t test. <b>C</b>, cyclic GMP content determined in renal cortical slices of sham- and 2K1C-operated WT mice without any addition (15 slices of 5 mice per group) or in the presence of sildenafil (100 µM, 10 min; 15 slices of 5 mice per group). ***P< 0.001 versus WT-sham, unpaired Student's t test. Experiments were performed using the non-clipped kidney of 2K1C mice and the respective kidney of sham mice.</p

Sildenafil effects on blood pressures.

<p>Sildenafil (100 mg/kg d) effects on systolic blood pressures measured in conscious sham- and 2K1C-operated WT (WT-sham 118 ± 1 mmHg without, and 112 ± 1 mmHg with sildenafil; WT-2K1C 130 ± 4 mmHg without, and 118 ± 2 mmHg with sildenafil) and NO-GC1 KO mice (KO-sham 118 ± 1 mmHg without, and 113 ± 2 mmHg with sildenafil; KO-2K1C 125 ± 2 mmHg without, and 121 ± 2 mmHg with sildenafil) using tail-cuff manometer (n=4 WT-sham, 5 WT-2K1C, 6 KO-sham, and 10 KO-2K1C mice). Sildenafil administration and SBP measurements were performed during the 4^th week after operation. *P< 0.05 2K1C WT compared to sham WT, unpaired Student's t test. SBP indicates systolic blood pressure. </p

Reduced cGMP response in kidneys of NO-GC1 KO mice.

<p><b>A</b>, NO-stimulated cGMP-forming activity (100 µM DEA-NO) determined in kidney homogenates of WT and NO-GC1 KO mice (n=19 and 6 mice, respectively). PDE activity in WT and KO kidneys measured with 1 µM cGMP as substrate (n=11 and 7 mice, respectively). ** P< 0.01 versus WT, unpaired Student's t test. B, absent of the α1 subunit in NO-GC1 KO kidney homogenates and quantification of the α2 and β1 subunit content with respect to the subunit amount in WT using subunit specific antibodies (n=8 mice per genotype). Representative strips with α1, α2, β1 and the respective β tubulin bands of the same lanes are shown above. *** P< 0.001 versus WT, unpaired Student's t test. C, cyclic GMP content determined in renal cortical slices of WT and KO mice without any addition (n=20 and 10 mice, respectively), or in the presence of ODQ (20 µM, 15 min; n=5 and 4 mice, respectively) or carbachol (30 µM, 3 min; n=10 and 6 mice, respectively) or DEA-NO (100 µM 3 min; n=10 and 7 mice, respectively). # P< 0.01 versus untreated slices in the same group; *P< 0.01 versus WT, unpaired Student's t test. D, carbachol-induced relaxation of NA-contracted isolated perfused kidneys of NO-GC1 KO (n=12) and WT mice (n=21). ** P< 0.01 versus WT, unpaired Student's t test. E, concentration-response curves for GSNO-induced relaxation of NA-contracted isolated perfused kidneys in the presence of L-NAME (n=5 WT and 7 KO mice). The white dots indicate the Cch-induced relaxation plotted on the curves to determine the corresponding GSNO concentration. F, Western blot detection of eNOS in 50 µg kidney homogenates of NO-GC1 KO (n=8) and WT mice (n=5) and quantification with respect to the eNOS amount in WT kidneys. A representative strip with eNOS and the respective β tubulin bands of the same lanes is shown above. * P< 0.05 versus WT, unpaired Student's t test.</p

2K1C operation did not up-regulate PDE5.

<p><b>A</b>, PDE5 mRNA in preglomerular vessels of sham- and 2K1C-operated WT mice detected by quantitative real-time PCR and quantified relative to GAPDH with the ∆C_T method (n=8 and 9 mice, respectively). <b>B</b>, PDE5 protein detected by Western blot in 50 µg kidney homogenates of sham- and 2K1C-operated WT mice and expressed as % of PDE5 content in kidneys of sham-WT mice (n=7 mice per group). A representative strip with PDE5 and the respective β tubulin bands of the same lanes is shown below. PDE activities in homogenates of kidneys and preglomerular vessels of sham- and 2K1C-operated WT mice in the absence and presence of sildenafil (100 nM) measured by 1 µM (<b>C</b>) or 30 nM cGMP (<b>D</b>). PDE activities were obtained of 3 mice per group in kidneys by 1 µM cGMP; n=13 and 10 mice per group, preglomerular vessels; n=6 per group kidneys by 30 nM. * P< 0.001 versus corresponding PDE activity without sildenafil, paired 2-tailed Student's t test. Experiments were performed using the non-clipped kidney of 2K1C mice and the respective kidney of sham mice.</p

2K1C operation did not reduce vascular relaxation in NO-GC1 KOs but in WT mice.

<p><b>A</b>, endothelium-dependent relaxation induced by carbachol (30 µM) in NA-contracted aortic rings and isolated perfused kidneys of sham- and 2K1C-operated WT mice (36 aortic rings of n=9 mice per group; kidneys of n=21 and 8 mice, respectively). * P< 0.05 versus aorta of WT-sham, paired 2-tailed Student's t test; ** P<0.001 versus kidney of WT-sham, unpaired 2-tailed Student's t test. <b>B</b>, concentration-response curves for GSNO-induced relaxation determined in NA-contracted isolated perfused kidneys of sham- and 2K1C-operated WT mice in the presence of L-NAME (n=5 and 7 mice, respectively). P=0.037, ANOVA for repeated measurements. <b>C</b>, endothelium-dependent relaxation induced by carbachol (30 µM) in NA-contracted aortic rings and isolated perfused kidneys of sham- and 2K1C-operated NO-GC1 KO mice (28 aortic rings of n=7 mice per group; kidneys n=12 and 7 mice, respectively). <b>D</b>, concentration-response curves for GSNO-induced relaxation determined in NA-contracted isolated perfused kidneys of sham- and 2K1C-operated NO-GC1 KO mice in the presence of L-NAME (n=8 and 6 mice, respectively). Experiments were performed using the non-clipped kidney of 2K1C mice and the respective kidney of sham mice.</p

Circulating NOS3 Modulates Left Ventricular Remodeling following Reperfused Myocardial Infarction

<div><p>Purpose</p><p>Nitric oxide (NO) is constitutively produced and released from the endothelium and several blood cell types by the isoform 3 of the NO synthase (NOS3). We have shown that NO protects against myocardial ischemia/reperfusion (I/R) injury and that depletion of circulating NOS3 increases within 24h of ischemia/reperfusion the size of myocardial infarction (MI) in chimeric mice devoid of circulating NOS3. In the current study we hypothesized that circulating NOS3 also affects remodeling of the left ventricle following reperfused MI.</p><p>Methods</p><p>To analyze the role of circulating NOS3 we transplanted bone marrow of NOS3^−/− and wild type (WT) mice into WT mice, producing chimerae expressing NOS3 only in vascular endothelium (BC−/EC+) or in both, blood cells and vascular endothelium (BC+/EC+). Both groups underwent 60 min of coronary occlusion in a closed-chest model of reperfused MI. During the 3 weeks post MI, structural and functional LV remodeling was serially assessed (24h, 4d, 1w, 2w and 3w) by echocardiography. At 72 hours post MI, gene expression of several extracellular matrix (ECM) modifying molecules was determined by quantitative RT-PCR analysis. At 3 weeks post MI, hemodynamics were obtained by pressure catheter, scar size and collagen content were quantified post mortem by Gomori’s One-step trichrome staining.</p><p>Results</p><p>Three weeks post MI, LV end-systolic (53.2±5.9μl;***p≤0.001;n = 5) and end-diastolic volumes (82.7±5.6μl;*p<0.05;n = 5) were significantly increased in BC−/EC+, along with decreased LV developed pressure (67.5±1.8mmHg;n = 18;***p≤0.001) and increased scar size/left ventricle (19.5±1.5%;n = 13;**p≤0.01) compared to BC+/EC+ (ESV:35.6±2.2μl; EDV:69.1±2.6μl n = 8; LVDP:83.2±3.2mmHg;n = 24;scar size/LV13.8±0.7%;n = 16). Myocardial scar of BC−/EC+ was characterized by increased total collagen content (20.2±0.8%;n = 13;***p≤0.001) compared to BC+/EC+ (15.9±0.5;n = 16), and increased collagen type I and III subtypes.</p><p>Conclusion</p><p>Circulating NOS3 ameliorates maladaptive left ventricular remodeling following reperfused myocardial infarction.</p></div

Flow chart of the presented study.

<p>In a closed chest model, animals were subjected to reperfused myocardial infarction. After 60 min of ischemia, animals were divided into two different groups: 1) 72 h post MI 2) 3 weeks post MI. Further analysis followed as depicted.</p

BC−/EC+ exhibited increased end-systolic and end-diastolic volume and decreased left ventricular function 3 weeks post MI.

<p>BC−/EC+ exhibited an increase in end-systolic (<b>A</b>) and end-diastolic volume (<b>B</b>), a significantly more pronounced decrease in stroke volume (<b>C</b>) (BC+/EC+ n = 8 and BC−/EC+ n = 5; two-way ANOVA and Bonferroni’s post hoc test or student’s t-test; * p<0.05, ** p≤ 0.01 BC+/EC+ vs. BC−/EC+; # p<0.05, ## p≤ 0.01, ### p≤ 0.001 BC+/EC+ at different time points; $p<0.05,$$p≤ 0.01,$ $$ p≤ 0.001 BC−/EC+ at different time points), and decreased left ventricular developed pressure (<b>D</b>) 3 weeks post MI compared to BC+/EC+ (BC+/EC+ n = 24 and BC−/EC+ n = 18; student‘s t-test; ***p≤0.001).</p

Table_1_On the Effects of Reactive Oxygen Species and Nitric Oxide on Red Blood Cell Deformability.docx

<p>The main function of red blood cells (RBCs) is the transport of respiratory gases along the vascular tree. To fulfill their task, RBCs are able to elastically deform in response to mechanical forces and, pass through the narrow vessels of the microcirculation. Decreased RBC deformability was observed in pathological conditions linked to increased oxidative stress or decreased nitric oxide (NO) bioavailability, like hypertension. Treatments with oxidants and with NO were shown to affect RBC deformability ex vivo, but the mechanisms underpinning these effects are unknown. In this study we investigate whether changes in intracellular redox status/oxidative stress or nitrosation reactions induced by reactive oxygen species (ROS) or NO may affect RBC deformability. In a case-control study comparing RBCs from healthy and hypertensive participants, we found that RBC deformability was decreased, and levels of ROS were increased in RBCs from hypertensive patients as compared to RBCs from aged-matched healthy controls, while NO levels in RBCs were not significantly different. To study the effects of oxidants on RBC redox state and deformability, RBCs from healthy volunteers were treated with increasing concentrations of tert-butylhydroperoxide (t-BuOOH). We found that high concentrations of t-BuOOH (≥ 1 mM) significantly decreased the GSH/GSSG ratio in RBCs, decreased RBC deformability and increased blood bulk viscosity. Moreover, RBCs from Nrf2 knockout (KO) mice, a strain genetically deficient in a number of antioxidant/reducing enzymes, were more susceptible to t-BuOOH-induced impairment in RBC deformability as compared to wild type (WT) mice. To study the role of NO in RBC deformability we treated RBC suspensions from human volunteers with NO donors and nitrosothiols and analyzed deformability of RBCs from mice lacking the endothelial NO synthase (eNOS). We found that NO donors induced S-nitrosation of the cytoskeletal protein spectrin, but did not affect human RBC deformability or blood bulk viscosity; moreover, under unstressed conditions RBCs from eNOS KO mice showed fully preserved RBC deformability as compared to WT mice. Pre-treatment of human RBCs with nitrosothiols rescued t-BuOOH-mediated loss of RBC deformability. Taken together, these findings suggest that NO does not affect RBC deformability per se, but preserves RBC deformability in conditions of oxidative stress.</p